Method and device for the concentration of multiple microorganisms and toxins from large liquid toxins

ABSTRACT

A method for the simultaneous concentration of multiple toxins from large volumes of water. The method includes the steps of providing a disposable separation centrifuge bowl, the centrifuge bowl including a positively charged material at it&#39;s inner core. A large water sample contaminated with toxins from a group consisting of protozoa, bacteria, bacterial spores, and toxins is delivered to the centrifuge bowl. A centrifugal force is applied to the separation bowl. The water sample is concentrated to remove large particles of the toxins in the bowl due to the centrifugal forces. The concentrated water sample is passes through the positively charged inner core to capture any remaining concentrated targets by electrostatic forces and the concentrated targets are eluted.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Stage of InternationalApplication No. PCT/US2008/066894 filed on Jun. 13, 2008, whichdesignates the United States, and which claims the benefit of priorityunder 35 U.S.C. §119(e) of U.S. provisional application No. 60/934,417,filed Jun. 13, 2007, the contents of which are incorporated herein byreference in its entirety.

GOVERNMENT SUPPORT

This invention was made with United States Government support underGrant No. RD83300301 awarded by the Environmental Protection Agency, andGrant No. W911NF-07-C-0030 awarded by the Army Research Office. TheUnited States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and device for thesimultaneous concentration of multiple waterborne pathogens from largevolumes of liquid toxins, and more particularly to the effectiveconcentration of small numbers of multiple waterborne pathogens fromlarge volumes of various water matrices.

2. Description of the Related Art

Effective concentration of small numbers of multiple waterborn pathogensfrom large volumes of various water matrices is a serious challenge.Current commercially available technologies are not able toautomatically concentrate and elute simultaneously multiple pathogensfrom large liquid volumes using portable equipment. Pathogens size,variability and biological differences requires the use of specific,expensive, and labor intensive concentration techniques, especially forviruses and protozoa.

Currently, the two major approaches for concentrating multiplemicroorganisms from large water volumes include filtration andcontinuous flow centrifugation. Ultrafilters, which organisms areretained by size exclusion have much smaller pore size than viruses,which allow for the simultaneous concentration of viruses, bacteria, andprotozoa. The particles are kept in the retentate and thus preventclogging of the filter surface. Several publications have described theapplication of hollow fiber ultrafilters for concentrating humanviruses, see Bicknell, 1985, Cryptosporidium oocysts (Simmons, 2001;Kuhn and Oshima, 2002, Ferguson, 2004), and the simultaneousconcentration of viruses, bacteria and protozoa (Juliano and Sobsey,1997/1998; Morales-Morales, 2003; Hill 2005).

However, ultrafiltration methodology is cumbersome and requires furthersystematic evaluation for much larger volumes of water than the 100 Lvolumes that are currently available. Electropositive filters are notrated by pore size and were initially designed to capture viruses fromlarge water volumes by electrostatic attraction. There have been a fewpublications that evaluated their efficacy to concentrate bacteria andprotozoa. See Hou et al (1980) which reported substantially highretention of E. coli, Viruses (Polio, and MS2 bactenophages, andendotoxin) by using charged modified filters. Watt et al. (2002)reported low recoveries for Cryptosporidium oocysts, Giardia cysts andPolio virus from large water volumes, 14% and 17%, respectively.

Recently, two configurations of the ZetaPlus® Virosorb® 1 MDS filterwere evaluated for simultaneous recovery of multiple microorganism typesfrom tap water (Polaczky et al., 2007). High numbers of C. parvum, S.enterica and B. globigii, and bacteriophages, were spiked into 25 L oftap water samples. Low recoveries were achieved using the cartridgefilter, 21%, 37% and 87%, respectively and much higher recoveries, 28%,50%, 65%, respectively, were reported for the flat filter.

Continuous flow centrifugation (CFC) enables large scale collection ofparticles, such as protozoa and bacteria, through their sedimentationdue to high centrifugal forces. Whitmore and Carrington, (1993) employeda bench-top centrifuge for the recovery of C. parvum oocysts from watersamples.

Utilization of stationary bench-top blood cell separators withrecoveries of several folds higher than the cartridge filtration wasalso reported (Goatcher, 1996). Substantially high recoveries (>90%)were reported for simultaneous concentration of Cryptosporidium,Giardia, Microsporidium, and bacteria from small volumes of source andpotable water using a stationary centrifuge and a costly labor-intensiveprotocol (Borchardt and Spencer 1 996; Borchardt and Spencer, 1998;Borchardt and Spence 2002).

However, less efficient recoveries of C. parvum oocysts from 100 L werealso reported by this method (Swales and Wright, 2000). In addition,very low recoveries, less than 5%) of C. parvum from 10 L of waterspiked source water, using a compact continuous flow centrifuge werereported (Higgins, 2003).

The above reports demonstrate that currently suggested approaches forconcentrating multiple microbe types from large water samples arelimited by manually operated systems, using expensive disposables andemploying tedious protocols that are not suitable for rapid response toaccidental or deliberate water contamination and may complicate routinemonitoring.

An earlier prototype of a modified blood separation apparatus, such asthe 625B standard disposable bowl manufactured by Haemonetics, ofBraintree, Mass.) efficiently concentrated Cryptosporidium oocysts,Giardia cysts, and Microsporidian spores from tap and source watersamples. The basic principle underlying the Continuous FlowCentrifugation (CFC) technique has also been described (Zuckerman et al,1999; Zuckerman and Tzipori, 2004; 2006).

However, there still exists the need for a continuous flow centrifugethat enables concentration and elution of targets from large volumes ofliquids.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide an automated portablecontinuous flow centrifuge that enables complete automation of theconcentration and elution of the targets from large volumes of liquids.

Immediate and anticipated uses and/or commercial applicability of thecontinuous flow centrifuge of the present invention include: thesimultaneous concentration of viruses, protozoa, bacteria, bacterialspores, algae, and toxins from large volumes of liquids such as water,wastewater, milk, wine, beverages and other liquids; on line monitoringof microbial quality of liquids; and on line monitoring of biothreatagents in liquids

The device is simple to operate, rapid, robust and portable. The devicecan also be easily upscaled to concentrate larger volumes. The processof sample concentration and elution is completely automated and theresulting concentrate is small in volume and presentable to a variety ofdetection methods.

In accomplishing these and other aspects of the present invention, thereis provided a method for the simultaneous concentration of multipletoxins from large volumes of water. The method includes the steps ofproviding a disposable separation centrifuge bowl, the centrifuge bowlincluding a positively charged material at it's inner core. A largewater sample contaminated with toxins from a group consisting ofprotozoa, bacteria, bacterial spores, and toxins is delivered to thecentrifuge bowl. A centrifugal force is applied to the separation bowl.The water sample is concentrated to remove large particles of the toxinsin the bowl due to the centrifugal forces. The concentrated water sampleis passes through the positively charged inner core to capture anyremaining concentrated targets by electrostatic forces and theconcentrated targets are eluted.

There is further provided a device for concentrating multiple pathogensfrom large liquid samples. The device includes a centrifuge bowl and acore including a positively charged material located within thecentrifuge bowl. An inlet located at one end of the bowl receives theliquid sample containing the pathogens. The core has a passageway andthe liquid sample flows through the core and the concentrated pathogensare attracted and adhere to the positively charge material.

These and other objects, features, aspects, and advantages of thepresent invention will become more apparent from the following detaileddescription of the preferred embodiment relative to the accompanieddrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the portable concentrator of the presentinvention.

FIG. 2 is a perspective view of the top deck of the concentratorhousing.

FIG. 3 is a perspective view of the rear panel of the concentratorhousing.

FIG. 4 is a perspective view of the pump of the concentrator.

FIG. 5 is a perspective view of the large pneumatic valve of theconcentrator.

FIG. 6 is a perspective interior view of the concentrator of the presentinvention.

FIG. 7 is a perspective view of a partially cut away high separationbowl of the present invention.

FIG. 8 is an outer view of the core of the HS separation bowl of thepresent invention.

FIGS. 9A and 9B are cross-sectional views of the inner and outer coresof the HS separation bowl of the present invention.

FIG. 10 is a top view of the loading diagram of the disposable set.

FIG. 11 is a flow diagram of the operation of the concentrator of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The automated portable concentrator of the present inventionsimultaneously concentrates protozoa, bacteria, bacterial spores, andtoxins from large volumes of liquids. After completion of theconcentration cycle it elutes the concentrated targets and delivers theconcentrate to disposable sterile bags that can be presented in thefield to various detection kits or can be sent to a lab for analysis.The concentrate could also be presented to a variety of detectionmethods.

As will be described in further detail herein, the portable continuousflow device includes a disposable bowl that simultaneously concentratesmultiple pathogens from the same sample, and an automated portablecontinuous flow centrifuge that accommodates the modified bowl and adisposable tubing harness and allows for sample concentration andelution from large water volumes.

Referring to FIG. 1, the CFC100A™ concentrator 10 houses a centrifuge12, such as that made by Magstar Technologies of (Minneapolis, Minn.)and which is capable of running from 0 to 12,000 rpm (max 400 RCF), aperistaltic pump 14 (0-1000 ml/min), various high pressure pneumaticvalves 16, 18 and electronics that are all controlled by a PLC computersystem (FIG. 6) enclosed in a housing 20. Display housing 20 is compactand light, measuring, for example, 12×13×16 inches tall and weighing 30pounds. The PLC system has a preset protocol for 10 L sample volumes,but can be programmed for larger volumes. It is also capable of storingnumerous different protocols giving the machine a great deal ofversatility and flexibility. The power source can be a versatile 240/110VAC and there is an optional transport and storage cart which houses arechargeable battery pack to run the machine with DC power. A cover 22is hinged to top deck 24. The housing 20 and cover 22 and can be madefrom a 5051 aluminum or other suitable material. It should beappreciated that the described dimensions of the housing and theoperating parameters of the components are examples only and each can beadjusted depending upon the applications of the present invention.

Housing 20 includes a base 21 and a top deck 24 to which centrifuge 12and pump 14 are mounted. Motor control units (not shown) to control thecentrifuge and pump speeds are also mounted within the base. Pluralitiesof pinch valves 16, 18 are also mounted on the top deck. For example,two large valves 18 and three small valves 16 are provided to controlthe tubing flow. The PLC control and pneumatic module for the pinchvalves can also be mounted to the inside of the top deck.

As shown in FIG. 2, top deck 24 includes a plurality of apertures 25-27for the centrifuge, valves, and pump. A seal (not shown) can be placedaround the hole 25 in the top deck for the centrifuge and at the backbetween top deck 24 and cover 22 to prevent against water leakage intothe concentrator. Base 21 also includes a rear panel 28 as shown in FIG.3. Rear panel 28 has three sides that complete the base of the housing.A cooling fan 30 is mounted to the rear panels. A power entry module canalso be mounted at 32 and recessed handles 34 are also located on thepanel sides.

Referring to FIG. 4, pump 14 is used to control the direction and flowrates of fluids during the concentration and elution procedures. Pump 14can include for example, three pump rollers that are spring loaded withthree springs per roller and slide in a miter groove. Such aconfiguration is sufficient for a ⅜″ tubing. Pump 14 includes a rotor 36that is driven by a pump motor 36 using a mating square configurationbetween the motor shaft and the rotor. A shaft of the motor is attachedto the rotor by a threaded cap. Pump 14 includes a pump platen 40 thatpivots at one end in nylon bushings to allow tubing to be threadedthrough the pump. Rotor 36 and platen 38 are designed for high volumeblood processing. The pump motor can be a brushless stepper that allowsflow rates, for example, between 1 LPM to 0. A rotor guard 42 is fixedto the base 21 and encloses the rotor to protect the operator fromrotating parts. A platen sensor arm 44 is located on the platen shaft.If the platen is opened during pump operation the pump will come to astop.

As described above, a plurality of pneumatic valves 16, 18 are provided.Referring to FIG. 5, large pneumatic valve 18 includes a valve housing46 and a valve assembly 48. Both are attached to the underside of topdeck 24. Valve assembly 48 is located within valve housing 46. O-ringscan be provided to prevent leakage. Air pressure keeps the valve openwhile spring pressure keeps it close. Valve housing 46 can be made ofhard coated 6061 aluminum or another appropriate material. Smallpneumatic valves 16 can be a one-piece assembly, for example, those madeby Aerodyne Controls of Ronkonkoma, N.Y.

Referring to FIG. 6, the concentrator 10 includes a PLC 50 locatedwithin base 21. PLC can have numerous inputs, outputs and communicationports used for programming, operation interfaces or networking, as isknown in the art and can be similar to the PLC manufactured byAutomation Direct of Atlanta, Ga. One communication port should bemulti-functional and capable of handling RS485, RS232C, and RS422communication links. Over 220 different instructions are available forprogram development, as well as, extensive internal diagnostics that canbe monitored from the application program or from an operator interface.Handheld programmers, operator interfaces or a personal computer can beeasily connected without the need for any additional hardware. The PLCcan be operated on 24 volt DC power. Also, PLC 50 can operate withBoolean ladder instruction programming that uses 16-bit register virtualmemory for data storage. Software, such as DL06PLC or DirectSOFT5 forWindows® installed on a personal computer cabled to the PLC. Programstorage can be via FLASH memory that is part of the CPU board in thePLC. In addition, RAM within the CPU can store system parameters,Virtual Memory and other data that is not in the application program.The RAM can be backed up by a super-capacitor, storing the data forseveral hours in the event of a power outage. The capacitor canautomatically recharge during powered operation of the PLC. A pneumaticsmodule 52 is also located within base 21. Pneumatics module 52 isdesigned to supply air pressure to pneumatic valves 16 and 18. The CFCcontains two motor controllers, one for the centrifuge and one for thepump.

The CFC is powered by and an Astrodyne MK75s-24, which has and inputvoltage range of 90-264 VAC and an output voltage of 25 VDC. A relay isused for communication between the PLC and the motor controllers and canbe a 70-782EL8-1 socket relay, manufactured by Magnacraft of Milwaukee,Wis., and is an eight pin logic style DIN panel with an elevatorterminal or module compatible relay. It has a normal voltage rating of300 volts and a normal current rating of 12 amps.

An EMI filter, such as that manufactured by Filter Concepts of SantaAna, Calif., is designed to effectively attenuate differential andcommon mode noise from switch mode power conversion and regulationcircuits subject to FCC Class B or CISPRJEN Class A conducted EMIlimits. Maximum attenuation is achieved at 150 kHz and above. The filteris also available with an A-circuit option to enhance its performance inapplications with high source impedance.

The present invention also provides for a sterile, disposable set 58(FIG. 10) that includes a high separation core bowl, PVC tubing and atleast two collection bags required for the concentrated sample's elute.The disposable set is packaged separately in a sealed bag and ETOsterilized. Referring to FIG. 7, a high separation disposable bowl 60includes a bowl 62 and a two-piece core 64, which will be describedfurther herein, located within bowl 62. HS bowl 60 is designed to beplaced within centrifuge 12. At one end bowl 62 is a basin 66 in which,protozoa, bacteria, and other large particles are compacted and fromwhich virus buffer is collected depending upon the stage of operation ofthe concentrator. Located at the other end of bowl 62 is an inlet port68 through which a water sample is delivered to the HS bowl. An outletport 70 is also located at the end of the bowl through which virusbuffer is injected during a certain stage of operation. An inner fluidpassageway 67 travels through the core.

Referring to FIGS. 7 and 8, the two-piece core 64 is a modification of acore manufactured by Heaomonetics. The core includes a plurality ofholes 72 drilled around the lower perimeter of the core to allow for ahomogeneous flow of water through charged material of the core, whichwill be described herein. For example, six sets of three holes at 45degree increments can be drilled around the lower perimeter, for a totalof 24 holes. As shown in FIGS. 9A and 9B, core 64 includes an outer core74 and an inner core 78. Located between cores 74 and 78 is a layer ofpositively charged filter material 76. Charged filter material 76 willbe further described herein.

As mentioned above, the portable equipment uses disposable set 58 thatuses ⅜″ outside diameter by ¼″ inside diameter large tubing, 3/16″outside diameter by ⅛ inside diameter small tubing, bowl 60 and twoplastic bags (100 ml for the viruses and 400 ml for theprotozoa/bacteria). During the concentration process, the large diametertubing delivers the liquid samples to the pump and into the disposablebowl. The large particles (protozoa, bacteria, spores, suspendedmaterial and debris) are sedimented, the “cleaner” liquid which containsnegatively charged viruses/toxins is forced through the core and virusesand toxins are captured by strong electrical force. Then, the largetubing directs the waste water from the machine. During the automatedelution process, the small tubing directs the protozoa/bacteria bufferinto the bowls residual and also delivers the virus buffer into thecore. The large bag initially accommodates the protozoa/bacteria bufferand after the elution it contains the protozoa/bacteria concentrate. Thesmall bag contains the virus buffer and retains the virus concentrate.

Referring to FIG. 10, the large diameter tubing is attached to the inletand outlet ports of the HS core bowl, which is held in the centrifugechuck. As shown, the pump drives the water sample through the largediameter tubing to the inlet port of the bowl. Waste water is carriedthrough large diameter tubing from the outlet port of the bowl. Duringthe elution process the small tubing carries the elution buffers fromthe bags to the centrifuge bowl and carries the concentrate eluate backto the bags when the elution cycle concludes. The set is coupled by avariety of PVC connectors. The high separation core bowl 60 includespositively charged material and a membrane welded to the top to retainthe material.

As described above, disposable continuous flow centrifuge bowl 60 has apositive charged matrix for virus capturing, simultaneously withbacteria, protozoa and toxins. The positive charged core allows forsimultaneous concentration of protozoa, bacteria, spores, toxins andviruses, from large water volumes.

Two different techniques were used to prepare the charged component. Thefirst configuration was prepared as follows: the outer glass fibercovers of a 12″×9.5″ sheet of ViroCap viral filter manufactured byArgonide Co. of Sanford, Fla., were gently peeled away, removing themfrom the inner membrane filter. This inner membrane filter was then cutinto strips, placed into a blender, and shred to a consistency of“cotton”. The same was repeated with an equal volume of tissue paper. Anequal weight of 15 grams each of ViroCap viral filter “cotton”, andtissue “cotton” were then added to one bag containing 10 grams ofaluminum hydroxide Al(OH)₃ nano-ceramic fibers Boehmite R0608 byArgonide Co. The bag was then sealed and mixed thoroughly, so as toevenly distribute all materials.

The out flow chamber of the core was then packed with the final productof the filter material prep. To further ensure a homogenous mix, thevolume of filter material prep for each core, approximately 4 grams,were teased apart and mixed manually before being inserted into thecore. A 6″ thin plastic ruler was used to pack the prepped material intothe core out flow chamber, a consistent and form an optimal homogenous,dense but fluffy filter matrix. A fiber cover support, for example,Hollytex 3267 spunbond polyester, Haemonetics part #50996-00, made byAhlstrom Technical Specialties of Mt. Holly Springs, Pa., was cut to fitto seal the top of the out flow chamber, securing the filter materialprep within the out flow chamber.

A second configuration involved the preparation of a slurry composed of1 g Pyrex fiber glass wool, 8 micron pore size, borosilicate such asAldrich CLS3950 manufactured by Sigma of St. Louis, Mo., 1 g ofnano-ceramic Boehmite fibers powder, for example, R0608 of ArgonideCorp., of Sanford, Fla.) and 1 g of cellulose microcrystalline powder(Aldrich 435236 by Sigma), 1 g of paper pulp, and 5 ml of DI water whichwas mixed thoroughly, and was poured into the inner space of the bowl'sinsert. The insert was then placed in a dry oven overnight at 70° C.Both types of modified cores were then sent to Haemonetics and used inthe final assembly of the HS plasma aphaeresis bowls.

The present invention also encompasses a continuous flow centrifugationmethodology which allows for the simultaneous concentration andprocessing of protozoa, bacteria, viruses, and toxins presented in largewater volumes. The overall process involves the following steps: a)concentration; and b) elution. In the concentration step, theperistaltic pump delivers a water sample through the bowl's inlet port68, the large particles such as protozoa, bacterial spores/cells andother suspended material is sedimented due to the strong centrifugalforces and the much cleaner water sample is forced to flow through thedesignated passages in the bottom of the inner core, where as thenegatively charged viruses are captured by the strong electrostaticattraction, eliminates clogging issues that are common in standardfiltration components.

During elution protozoa/bacteria are dislodged by the addition ofdetergent and agitation, and the viruses which are trapped in the virusmatrix are released using a protein buffer which inverts the negativecharge. Both the virus and protozoa/bacteria concentrates are deliveredto a sterile bag. The bags could be processed in the field by usingportable detection kits or delivered to the laboratory.

Referring to FIGS. 10 and 11, the operation of the concentrator of thepresent invention will be described. The concentrator is started and thedisplay panel 54 (FIG. 1) will read “Press Select.” When the selectbutton is pressed all of the valves 16, 18 are opened. The centrifugedoor is opened. A new, sterile disposable set 58 is opened and removedfrom its packaging. The HS disposable bowl 60 is inserted intocentrifuge 12 and the centrifuge door is closed and locked. Next, thelarge diameter inlet tube is connected to the inlet port of the bowl andthread through the pump leaving the pump stop on the outside of theguard. The pump platen is closed and the inlet tube is pressed intovalve 1. The large diameter outlet tube is then connected to the outletport of the bowl and press fitted into valve 5. The bags containing theelution buffers on hooks on the cover. The tubing connected to thebacteria elution bag is pressed into pinch valve 2. The remaining tubingis press-fitted into valves 3 and 4. The select button on the display ispressed to close all the valves. The slide clamps on the tube of eachbag containing the elution buffers are then opened. To beginconcentration, the inlet tube is placed into the water sample and theoutlet tube is positioned within the waste container. The start buttonon the display panel is pressed to begin automatic concentration andelution of the water sample. Once the entire concentration and elutionprocess is complete the display panel will read “Cycle Complete.” Theslide clamps on each bag are closed and the bags are removed and keptfor analysis. The remainder of the set is discarded.

Currently, a system which simultaneously concentrates and recoversmultiple types of microorganisms from large water volumes is notcommercially available. A continuous flow centrifugation methodologywhich allows for the simultaneous concentration and processing ofprotozoa, bacteria, and viruses presented in large water volumes wasdeveloped according to the present invention.

Several stages of experimentation led to the development of the system.In order to test the method recovery efficiency, Cryptosporidiumoocysts, Bacillus anthracis spores and MS2 bacteriophages weresimultaneously spiked in 10-50 L tap and turbid surface water samples,resulting in mean recoveries of 40%, 35%, and 50% respectively.

The automated portable concentrator was challenged with small numbers ofCryptosporidium oocysts, Bacillus anthracis spores and MS2bacteriophages simultaneously spiked in 10 L tap and turbid surfacewater samples, resulting in mean recoveries of 40%, 35%, and 50%respectively. The following is the summary of the results for 10 L oftap water.

TABLE 1 Recovery of C. parvum oocysts, MS2 bacteriophages and B.anthracis from 10 and 50 L tap water samples using a modified HS CoreBowl and a manually operated centrifuge. Spike Percent Spike PercentSpike Percent Vol. dose Recovery dose recovery dose recovery analyzedoocysts oocysts pfu/mL pfu/mL cfu cfu (L) (# (mean +/− (mean +/− (mean+/− (mean +/− (mean +/− (mean +/− replicates) SD) SD) SD) SD) SD) SD) 10(11) 100 +/− 39.7 +/− 2.1 * 10⁷ +/− 56.0 +/− 46.8 +/− 37.0 +/− 15.6 2.54.9 8.3 * 10⁶ 32.3 38.3 50 (3)  100 +/− 31.3 +/− 1.0 * 10⁷ +/− 71.1 +/−12.7 +/− 59.6 +/− 4.3 2.5 9.0 1.7 * 10⁷ 50.0 15.9

TABLE 2 Recovery of C. parvum oocysts, MS2 bacteriophage and B.anthracis from 10 L tap water samples using a modified HS Core Bowl andan automated centrifuge. Spike Percent Spike Percent Spike Percent Vol.dose Recovery dose recovery dose recovery analyzed oocysts oocystspfu/mL pfu/mL cfu cfu (L) (# (mean +/− (mean +/− (mean +/− (mean +/−(mean +/− (mean +/− replicates) SD) SD) SD) SD) SD) SD) 10 (7) 100 +/−40.0 +/− 2.6 * 10⁷ +/− 48.1 +/− 28.2 23.3 +/− 43.6 +/− 2.5 12.2 1.3 *10⁷ 4.6 16.4

Tap water samples were obtained from the Division of InfectiousDiseases, Cummings School of Vetinary Medicine. This tap water wassupplied by the North Grafton Water Utility and is initially collectedfrom a local ground water source. Turbidity of the sample was measuredat 0.002 NTU by a DRT-15CE Tubidimeter, such as that manufactured by HFScientific of Fort Meyers, Fla. Surface water samples were thencollected from a near-by pond. The samples were autoclaved and the meanturbidity post sterilization was measured at 3 NTU.

Ten liters of each water matrix was transferred to sterile, disposableplastic cubitainers. The cubitainer was placed on a stir plate andstirred while being seeded with an aliquot of each representativeorganism and refilled with none liters of water once one liter of theoriginal sample remained to be concentrated.

Prior to concentration the disposable harness is assembled in themachine according to the schematic layout of FIG. 10. Elution buffersare injected, for example, 5 ml of bacteria/protozoa and 20 ml of virus,into the designated bags using a Lauer lock syringe and the inlet tubingis placed into a 101 cubitainer containing the water sample. Referringto the flow chart of FIG. 11, the power source is turned on and therequired protocol is selected. During concentration of the water sample,pathogens are retained inside the centrifuge bowl at 9000 RPM. The watersample is driven through the centrifuge bowl while it is spinning at 0.5l/min by the peristaltic pump, which results in the retention of largerparticles of protozoa and bacteria on the wall of the disposablecentrifuge bowl. Small particles, such as virus, which escape thecentrifugal forces are forced through the positively charged filtermaterial in the out-flow chamber of the core and adsorbed to thematerial. After concentration of the water sample, the residual liquidin the bowl and the out-flow chamber of the core are subjected to anautomated elution procedure that produces two separate volumes ofbacteria/protozoa eluate, 300 ml, and virus eluate of 20 ml.

After concentration, the residual liquid is subject to a bacteria andprotozoa elution cycle. 5 ml of elution buffer is delivered through theinlet port of the bowl from the designated PVC bag. Thebacteria/protozoa elution buffer is a 5×PBS and 0.06% of a tween 80solution is used as a detergent to dislodge bacteria and protozoa thathave compacted to the walls of the bowl during the concentrationprocess.

Once the elution buffer has been added, the centrifuge spins to a speedof 7000 rpm and then is immediately braked until it comes to a completestop to dislodge the bacteria and protozoa. The cycle of spinning andbraking is repeated ten times with a 10 second interval between eachcycle. Upon completion of the final spin and brake cycle the 275 mLeluate is pumped from the inlet port of the bowl and returned to the PVCbag that originally contained the elution buffer. The centrifuge is thenspun at 9000 rpm for 1 minute to extract any residual liquid from theout-flow chamber of the core, which is subsequently pumped from theinlet to the rest of the eluate. The entire volume, approximately 300mL, is split into two separate volumes of 150 mL for analysis ofbacteria and protozoa.

Following the bacteria and protozoa elution cycle, 10 mL of viruselution buffer is pumped into the out-flow chamber of the core throughthe outlet port of the bowl from the designated PVC bag. The viruselution buffer is a beef extract, glycine and Tween 80 protein solution(Scientific Methods, Granger, Ind.) used to neutralize the positivecharge of the filter material inside of the core and dislodge the virusthat were adhered during the concentration process.

After addition of the elution buffer, the filter material inside thecore is saturated for 5 minutes at which point the centrifuge is spunfor 1 minute at 9000 rpm to extract the liquid from the out-flow chamberof the core into the bowl. A second volume of 10 mL of virus buffer ispumped into to out-flow chamber and the process is repeated. The cycleends by pumping the entire volume of virus eluate, approximately 20 mL,from the inlet port to the PVC bag that originally contained the viruselution buffer.

Certain microorganisms were used. C. parvum oocysts were chosen for useas representative protozoa. Water samples were spiked with one of twocommercially available products. EasySeed, produced by BiotechnologyFrontiers, LTD, NSW, Australia, consists of 100 flow-sorted; gammairradiated C. parvum oocysts and Giardia lamblia cysts with a cell countstandard deviation of less than 2.5 for each microorganism. Samples werespiked with EasySeed by decanting the contents of each vial asprescribed by the manufacturer. The second spiking suspension wasproduced by the Wisconsin State Laboratory of Hygiene, Madison, Wis.,and consists of 100 C parvum oocysts in 10 mL of reagent grade water and0.01% Tween 20 with a cell count standard deviation of less than 2.5.The contents of one 10 mL tube was poured into the water sample and thetube was rinsed with 2 mL of 0.01% Tween 20 PBST followed by two 2 mLrinses of reagent grade water seeded the water samples. Each rinse wasshaken vigorously for 30 seconds before being decanted into the watersample. Enumeration of the protozoa eluate from spiked water samples wasperformed as described by Method 1622 (USEPA, 2001). Dynabeadsanti-Cryptosporidium Kit (Invitrogen Dynal AS, Oslo, Norway) wasemployed for selective separation of oocysts from water sampleconcentrates using immunomagnetic separation according to themanufacturers instructions. Crypt-A-Gb fluorescent monoclonal antibodies(Waterborne, Inc., New Orleans, La.) were used against C. parvum oocystsfor detection and enumeration with a fluorescent microscope (OlympusOptical CO., Tokyo, Japan). The recovered numbers were multiplied by afactor of 2.

B. anthracis Sterne, a kanamycin resistant strain, was selected asrepresentative bacteria. This strain of B. anthracis was constructed bythe replacement of particular RNA coding sequences with an omegaelement, Q-kan, conferring kanamycin resistance. The stock suspension ofB. anthracis was kept at −80° C. Each week during testing a spikingsuspension was produced by diluting 100 μL of the stock suspension into50 mL of maximum recovery diluent (Oxoid Ltd., Basingstoke, Hampshire,England) to an estimated concentration of 30 colony-forming units (cfu)per mL. Water samples were spiked by pipetting 1 mL of the spikesuspension to the sample while stirring. The spike dose was quantitatedby pipetting 1 mL of the spiking suspension to 50 mL of reagent gradewater, vortexing and vacuum filtrating through a 0.45 μm, 47 mm membrane(Millipore, Billerica, Mass.). Post filtration, the membranes wereplaced on LB agar plates (Acros Organics, N.J.) containing kanamycin(Fisher Scientific, Fair Lawn, N.J.), inverted and incubated overnightat 37° C. After incubation, colony-forming units with the typicalappearance of B. anthracis, opaque grey and white formations, werecounted and recorded. To quantitate the recovery of B. anthracis fromspiked water samples, the bacteria eluate was vacuum filtrated andincubated in the same manner. The recovered numbers were multiplied by afactor of 2.

Eschericlzia coli ATCC 15597: E. coli (American Type Culture Collection,Manassas, Va.) was used as host bacteria for MS2 bacteriophage. Freezedried samples were purchased from ATCC, re-hydrated with 0.850 mL of LBbroth (Acros Organics, N.J.) and 1 50 mL of sterile glycerol, aliquotedto a volume of 50 μL and stored at −80° C. To propagate the bacteria one50 μL aliquot was diluted in 10 mL of LB broth and incubated overnightat 37° C. and 200 RPM on a shaking incubator. After incubation 1.5 mL ofsterile glycerol was added to the host bacteria broth, which wassubsequently aliquoted to a volume of 200 μL and stored at −80° C. forno longer than one month. To quantitate the recovery of MS2bacteriophage from spiked water samples and to enumerate the spike doseof phage LB agar plates were prepared with a field of actively growingE. coli to spot the phage on. Prior to preparation of the plates 800 μLof LB broth was added to one 200 μL aliquot of overnight grown E. coliand incubated for two hours at 37° C. After incubation, the hostbacteria broth was spread evenly over an LB agar plate and the excessaspirated off. Serial dilutions of phage were than spotted onto theplate immediately after the E. coli had air-dried.

Results:

TABLE 3.1 Recovery of B. anthracis from 10 L tap water samples using astandard HS Core Bowl and a manually operated centrifuge. Spike dosePercent Recovery Vol. analyzed cfu cfu (L) (# replicates) (mean +/− SD)(mean +/− SD) 10 (4)  98.5 +/− 0.7 30.9 +/− 8.9  10 (10) 58.4 +/− 9.630.9 +/− 10.8 10 (10) 10.3 +/− 0.4 44.8 +/− 11.9

TABLE 3.2 Recovery of C. parvum oocysts and B. anthracis from 10 L tapwater samples using a standard HS Core Bowl and an automated centrifuge.Spike dose Percent Recovery Spike dose Percent recovery Vol. analyzedoocysts oocysts cfu cfu (L) (# replicates) (mean +/− SD) (mean +/− SD)(mean +/− SD) (mean +/− SD) 10 (5) 100 +/− 2.5 36.0 +/− 15.2 14.8 +/−3.8 90.2 +/− 9.0

TABLE 3.3 Recovery of C. parvum oocysts, MS2 bacteriophages and B.anthracis from 10 and 50 L tap water samples using a modified HS CoreBowl and a manually operated centrifuge. Spike Percent Spike PercentSpike Percent Vol. dose Recovery dose recovery dose recovery analyzedoocysts oocysts pfu/mL pfu/mL cfu cfu (L) (# (mean +/− (mean +/− (mean+/− (mean +/− (mean +/− (mean +/− replicates) SD) SD) SD) SD) SD) SD) 10(11) 100 +/− 39.7 +/− 2.1 * 10⁷ +/− 56.0 +/− 46.8 +/− 37.0 +/− 15.6 2.54.9 8.3 * 10⁶ 32.3 38.3 50 (3)  100 +/− 31.3 +/− 1.0 * 10⁷ +/− 71.1 +/−12.7 +/− 59.6 +/− 4.3 2.5 9.0 1.7 * 10⁷ 50.0 15.9

TABLE 3.4 Recovery of C. parvum oocysts, MS2 bacteriophage and B.anthracis from 10 L tap water samples using a modified HS Core Bowl andan automated centrifuge. Spike Percent Spike Spike Percent Vol. doseRecovery dose Percent dose recovery analyzed oocysts oocysts pfu/mLrecovery cfu cfu (L) (# (mean +/− (mean +/− (mean +/− pfu/mL (mean +/−(mean +/− replicates) SD) SD) SD) (mean +/− SD) SD) SD) 10 (7) 100 +/−40.0 +/− 2.6 * 10⁷ +/− 48.1 +/− 28.2 23.3 +/− 43.6 +/− 2.5 12.2 1.3 *10⁷ 4.6 16.4

Although there have been major advancement in methodologies andequipment aimed at detecting waterborne pathogens, it is imperative thatno matter how good the detection method is, an efficient sampleconcentration is required due to the small numbers of the targetmicroorganisms. Moreover, most of the cutting edge detectiontechnologies utilize portable detection equipment for field testing, butlack of an efficient portable sample concentrator restricts its use.Among the several approaches for an efficient sample concentration,continuous flow centrifugation appears to be very promising.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. It ispreferred therefore, that the present invention be limited not by thespecific disclosure herein, but only by the appended claims.

What is claimed is:
 1. A method for the concentration of multiplepathogens from large volumes of liquids comprising the steps of:providing a disposable separation centrifuge bowl, the centrifuge bowlincluding a positively charged material at it's inner core; delivering aliquid sample contaminated with the pathogens to the centrifuge bowl;applying a centrifugal force to the separation bowl; concentrating theliquid sample to remove large particles of the pathogens in the bowl dueto the centrifugal forces; passing the concentrated liquid samplethrough the inner core to capture any remaining concentrated targets byelectrostatic forces; and eluting the concentrated targets.
 2. Themethod of claim 1, wherein the step of concentrating the multiplepathogens comprises simultaneously concentrating pathogens from a groupconsisting of protozoa, bacteria, bacterial spores, and toxins.
 3. Themethod of claim 1, wherein the step of eluting the concentrate comprisesdislodging the concentrated targets by adding a detergent and agitatingthe concentrate.
 4. The method of claim 3, wherein the step of elutingfurther comprises adding a protein buffer to the separation bowl toinvert a negative charge of the concentrated targets.
 5. The method ofclaim 4, further comprising the step of delivering the eluted targets todisposable sterile bags.
 6. The method of claim 5, further comprisingthe step of presenting the bags of eluted targets to various detectionkits.
 7. The method of claim 5, further comprising the step of sendingthe bags of eluted targets to a lab for analysis.
 8. The method of claim5, further comprising the steps of discarding the separation bowl andbags after analysis.
 9. A method for the simultaneous concentration ofmultiple toxins from large volumes of water comprising the steps of:providing a disposable separation centrifuge bowl, the centrifuge bowlincluding a positively charged material at it's inner core; delivering awater sample contaminated with toxins from a group consisting ofprotozoa, bacteria, bacterial spores, and toxins to the centrifuge bowl;applying a centrifugal force to the separation bowl; concentrating thewater sample to remove large particles of the toxins in the bowl due tothe centrifugal forces; passing the concentrated water sample throughthe positively charged inner core to capture any remaining concentratedtargets by electrostatic forces; and eluting the concentrated targets.10. The method of claim 9, wherein the step of eluting the concentratecomprises dislodging the concentrated targets by adding a detergent andagitating the concentrate.
 11. The method of claim 10, wherein the stepof eluting further comprises adding a protein buffer to the separationbowl to invert a negative charge of the concentrated targets.
 12. Themethod of claim 9, further comprising the step of delivering the elutedtargets to disposable sterile bags.
 13. The method of claim 12, furthercomprising the step of presenting the bags of eluted targets to variousdetection kits.
 14. The method of claim 12, further comprising the stepof sending the bags of eluted targets to a lab for analysis.
 15. Themethod of claim 12, further comprising the steps of discarding theseparation bowl and bags after analysis.